1. Field of the Invention.
The present invention relates generally to the cellular mechanisms and machinery involved in the glycosylation of proteins manufactured by the cell. More particularly, the present invention involves altering the glycosylation capabilities of a cell in order to control the structure of carbohydrate groups attached during glycosylation.
2. Description of Related Art.
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography.
During the last decade, numerous processes and procedures have been developed for genetically engineering cells in order to produce a wide variety of proteins and glycoproteins. These procedures involve utilizing recombinant DNA technology to prepare a vector which includes genetic material that codes for a specific protein or glycoprotein. Upon introduction of the vector into the host cell, the inserted genetic material instructs the host cell's biochemical machinery to manufacture a specific protein or glycoprotein.
Problems have been experienced with the production of glycoproteins by genetically engineering host cells. Glycoproteins are proteins having carbohydrate groups attached at various points along the protein's amino acid backbone. The carbohydrate groups are commonly attached to asparagine, serine or threonine. The genetic sequence introduced into the host cell usually includes instructions with respect to the amino acid sequence of the protein and the location and structure of the carbohydrate groups. Most of the cell lines which are commonly used as host cells are capable of following the vector's instructions with respect to preparing a protein having a specific amino acid sequence. However, many host cells are not capable of following instructions with respect to glycosylation of the protein. For example, E. coli is a common host cell used in producing a wide variety of proteins. However, E. coli does not contain the cellular glycosylation machinery required to attach carbohydrate groups to the proteins it manufactures.
Unlike E. coli, many other host cells do have varying capabilities with respect to protein glycosylation. However, even though these cells have glycosylation capabilities, the glycosylation machinery is not controlled by the recombinant DNA vector. Accordingly, the glycoprotein produced by such host cells may differ in carbohydrate structure from the natural glycoprotein coded for by the vector.(1, 2)
Chinese hamster ovary (CHO) cells are a standard cell line used commercially for the high yield expression of glycoproteins from vectors engineered through recombinant DNA technology. The protein sequence of the glycoprotein expressed by CHO comes from the DNA transinfected into the cell while the structure of the carbohydrate portion of the glycoprotein is determined by the cellular machinery of the CHO cells. While most glycoproteins normally contain a mixture of NeuAc-.alpha.-2,6Gal and NeuAc-.alpha.-2,3Gal linkages on their N-linked oligosaccharides, CHO cells only make asparagine linked carbohydrate chains with terminal sialic acids in the NeuAc-.alpha.-2,3Gal linkage.(1,2)
For example, erythropoietin is a glycoprotein naturally occurring in humans which has N-linked carbohydrate groups with both the NeuAc-.alpha.-2,6Gal and NeuAc-.alpha. -2,3Gal linkages. CHO cells which are genetically engineered to produce erythropoietin can only produce this protein with the NeuAc-.alpha.-2,3Gal linkages.(1) Although a number of mutant CHO cell lines have been developed which have altered capabilities for protein glycosylation,(3) they are not suitable for the production of glycoproteins intended for use in animals. Indeed, the carbohydrate groups produced by the cells are truncated, resulting in the rapid clearance of the recombinant glycoproteins from the blood followed by degradation. Thus, while the glycoproteins produced by these mutant CHO cell lines do display in vitro biological activity, they are inactive in vivo because of the rapid clearance from the blood stream.
From the above, it is apparent that there is a need to develop a process which can be used to alter the glycosylation machinery of host cells in order to control the structure of carbohydrates attached during glycosylation. Such a process for controlling host cell glycosylation would be useful not only in expressing glycoproteins which accurately mimic naturally occurring proteins, but would also be useful in preparing glycoproteins having selected altered carbohydrate structures for diagnostic and research uses.